载pVAX1/Tat PTD-endostatin壳聚糖纳米基因载体的构建与体外活性评价
|
摘要
目的构建载内皮抑素(Es)重组表达质粒的壳聚糖纳米基因载体并评价其抑制内皮细胞增殖的活性。方法选用真核表达质粒p VAX1为重组载体,采用PCR及双酶切法制备能够表达Tat PTD-Es融合蛋白的真核表达载体p VAX1/Tat PTD-Es;离子交联法制备壳聚糖载基因纳米粒,琼脂糖凝胶电泳筛选处方,检测纳米粒的形态、Zeta电位及粒径;采用最佳处方比例制备载重组质粒纳米载体,转染人脐静脉内皮细胞HUVEC,采用ELISA法测定分泌型Tat PTD-Es蛋白的表达量,CCK-8法测定对HUVEC的抑制作用,以上两个实验均以LipofectaminTM2000为阳性对照。结果 p VAX1/Tat PTD-Es的真核表达载体构建成功,壳聚糖纳米粒Zeta电位(14.3±1.6)m V,粒径(145±12)nm,纳米粒转染HUVEC后细胞上清中Tat PTD-Es的浓度为(182.5±16.3)ng/m L,壳聚糖基因载体转染HUVEC后对细胞有明显的抑制作用,72 h的抑制率达(53.4±5.4)%(Z=-2.611,P=0.008)。结论构建的壳聚糖纳米基因载体能够成功转染HUVEC并能明显地抑制内皮细胞的增殖。
Objective To develop chitosan gene nanoparticles loaded with recombinant plasmid which contains endostatin( Es) gene and evaluate its in vitro inhibitory effects on HUVEC cells. Methods p VAX1 was taken as the expression vector. PCR combined with double restriction enzyme digest method was used to construct recombinant plasmids p VAX1/Tat PTD-Es. Chitosan nanoparticles loaded with plasmids were prepared by ion cross-linking method. Agarose gel electrophoresis experiment was employed to screen the best prescription of nanoparticles. The shape,Zeta potential and size distribution of nanoparticles were evaluated. After nanoparticles were transferred into HUVEC cells,ELISA method was used to detect the expression of Tat PTD-Es,and CCK-8 was explored to determine the inhibitory effect on HUVEC. In ELISA and CCK-8 experiments,LipofectamineTM2000 loaded with p VAX1/Tat PTD-Es was used as positive control. Results The recombinant plasmid p VAX1/Tat PTD-Es was constructed successfully. The Zeta potential of chitosan nanoparticles loaded with recombinant plasmid was( 14. 3 ± 1. 6) m V,the size distribution was( 145 ± 12)nm. The concentration of Tat PTD-Es in HUVEC supernatant was( 182. 5 ± 16. 3) ng/m L. CCK-8 results showed that chitosan nanoparticles loaded with p VAX1/Tat PTD-Es had inhibitory effects on HUVEC cells and the inhibitory ratein 72 h was( 53. 4 ± 5. 4) %( Z =-2. 611,P = 0. 008). Conclusion Chitosan nanoparticles loaded with recombinant plasmid p VAX1/Tat PTD-Es can be transferred into HUVEC cells,and have distinct inhibitory effects on the proliferation of HUVEC cells.
Objective To develop chitosan gene nanoparticles loaded with recombinant plasmid which contains endostatin( Es) gene and evaluate its in vitro inhibitory effects on HUVEC cells. Methods p VAX1 was taken as the expression vector. PCR combined with double restriction enzyme digest method was used to construct recombinant plasmids p VAX1/Tat PTD-Es. Chitosan nanoparticles loaded with plasmids were prepared by ion cross-linking method. Agarose gel electrophoresis experiment was employed to screen the best prescription of nanoparticles. The shape,Zeta potential and size distribution of nanoparticles were evaluated. After nanoparticles were transferred into HUVEC cells,ELISA method was used to detect the expression of Tat PTD-Es,and CCK-8 was explored to determine the inhibitory effect on HUVEC. In ELISA and CCK-8 experiments,LipofectamineTM2000 loaded with p VAX1/Tat PTD-Es was used as positive control. Results The recombinant plasmid p VAX1/Tat PTD-Es was constructed successfully. The Zeta potential of chitosan nanoparticles loaded with recombinant plasmid was( 14. 3 ± 1. 6) m V,the size distribution was( 145 ± 12)nm. The concentration of Tat PTD-Es in HUVEC supernatant was( 182. 5 ± 16. 3) ng/m L. CCK-8 results showed that chitosan nanoparticles loaded with p VAX1/Tat PTD-Es had inhibitory effects on HUVEC cells and the inhibitory ratein 72 h was( 53. 4 ± 5. 4) %( Z =-2. 611,P = 0. 008). Conclusion Chitosan nanoparticles loaded with recombinant plasmid p VAX1/Tat PTD-Es can be transferred into HUVEC cells,and have distinct inhibitory effects on the proliferation of HUVEC cells.
引文
[1]OReilly MS,Boehm T,Shing Y,et al.Endostatin:an endogenous inhibitor of angiogenesis and tumor grow th[J].Cell,1997,88(2):277-285.
[2]王晓燕,袁中芳,魏增涛,等.重组IFN-l蛋白联合endostatin基因对碱烧伤诱导角膜新生血管的抑制作用[J].基础医学与临床,2005,25(12):1119-1123.WANG Xiaoyan,YUAN Zhongfang,WEI Zengtao,et al.Inhibitory effect of recombinant IFN-αcombined w ith endostatin gene on corneal neovascurization induced by alkaili burn[J].Basic and Clinical M edicine,2005,25(12):1119-1123.
[3]Mohajeri A,Pilehvar-Soltanahmadi Y,PourhassanM oghaddam M,et al.Cloning and expression of recombinant human endostatin in periplasm of Escherichia coli expression system[J].Adv Pharm Bull,2016,6(2):187-194.
[4]Tan H,Yang S,Feng Y,et al.Characterization and secondary structure analysis of endostatin covalently modified by polyethylene glycol and low molecular w eight heparin[J].J Biochem,2008,144(2):207-213.
[5]Fu W,Zhuo J,and Hu L.Differential effects of recombinant human endostatin treatment on differentiated and undifferentiated blood vessels in Lew is lung cancer[J].Oncol Lett,2017,13(1):196-200.
[6]Zhang X,Li Y,Cheng Y,et al.Tat PTD-endostatin:a novel anti-angiogenesis protein w ith ocular barrier permeability via eye-drops[J].Biochim Biophys Acta,2015,1850(6):1140-1149.
[7]Li Y,Li L,Li Z,et al.Tat PTD-Endostatin-RGD:a novel protein w ith anti-angiogenesis effect in retina via eye drops[J].Biochim Biophys Acta,2016,1860(10):2137-2147.
[8]张新科.穿膜肽Tat PTD介导的内皮抑素穿透眼球屏障及对眼部血管增生抑制作用的研究[D].济南:山东大学,2012.ZHANG Xinke.Study on the penetration of eye barrier and inhibition of ocular angiogenesis of endostatin w ith the mediation of Tat PTD[D].Jinan:Shandong University,2012.
[9]Campochiaro PA,Lauer AK,Sohn EH,et al.Lentiviral vector gene transfer of endostatin/angiostatin for macular degeneration(GEM)study[J].Hum Gene Ther,2017,28(1):99-111.
[10]Ojea-Jiménez I,Tort O,Lorenzo J,et al.Engineered nonviral nanocarriers for intracellular gene delivery applications[J].Biomed M ater,2012,7(5):054106.doi:10.1088/1748-6041/7/5/054106.
[11]Wang JJ,Zeng ZW,Xiao RZ,et al.Recent advances of chitosan nanoparticles as drug carriers[J].Int J Nanomedicine,2011,(6):765-774.
[12]Veilleux D,Nelea M,Biniecki K,et al.Preparation ofconcentrated chitosan/DNA nanoparticle formulations by lyophilization for gene delivery at clinically relevant dosages[J].J Pharm Sci,2016,105(1):88-96.
[13]Ramesan RM,Sharma CP.Modification of chitosan nanoparticles for improved gene delivery[J].Nanomedicine,2012,7(1):5-8.
[14]姚金凤,郭晨阳,杨红,等.壳聚糖纳米粒在药物输送中的应用研究进展[J].国际药学研究杂志,2013,40(1):90-94.YAO Jinfeng,GUO Chenyang,YANG Hong,et al.Application of chitosan nanoparicles for drug delivery:research advances[J].J Int Pharm Res,2013,40(1):90-94.
[15]Babu A,Ramesh R.Multifaceted applications of chitosan in cancer drug delivery and therapy[J].M ar Drugs,2017,15(4):96-104.
[16]Zhang X,Yang X,Ji J,et al.Tumor targeting strategies for chitosan-based nanoparticles[J].Colloids Surf B Biointerfaces,2016,148(1):460-473.
[17]Key J,Park K.Multicomponent,tumor-homing chitosan nanoparticles for cancer imaging and therapy[J].Int J M ol Sci,2017,18(3):594-612.
[18]Jung SP,Siegrist B,Hornick CA,et al.Effect of human recombinant endostatin protein on human angiogenesis[J].Angiogenesis,2002,5(1-2):111-118.
[19]Chen J,Liu DG,Yang G,et al.Endostar,a novel human recombinant endostatin,attenuates liver fibrosis in CCl4-induced mice[J].Exp Biol M ed,2014,239(8):998-1006.
[20]Ling Y,Yang Y,Lu N,et al.Endostar,a novel recombinant human endostatin,exerts antiangiogenic effect via blocking VEGF-induced tyrosine phosphorylation of KDR/Flk-1 of endothelial cells[J].Biochem Biophys Res Commun,2007,361(1):79-84.
[21]Xing P,Zhang J,Yan Z,et al.Recombined humanized endostatin(Endostar)combined w ith chemotherapy for advanced bone and soft tissue sarcomas in stage IV[J].Oncotarget,2017,8(22):36716-36727.
[22]Wigler M,Silverstein S,Lee LS,et al.Transfer of purified herpes virus thymidine kinase gene to cultured mouse cells[J].Cell,1977,11(1):223-232.
[23]张艳,马春红,孙汶生,等.高生物安全性人内皮抑素载体抑瘤作用的研究[J].中国现代普通外科进展,2004,7(6):350-355.ZHANG Yan,M A Chunhong,SUN Wensheng,et al.The antitumor effect of the high bio-safety vector containing human endosatin[J].Chin J Curr Adv Gen Surg,2004,7(6):350-355.
[24]Petkar KC,Chavhan SS,Agatonovik-Kustrin S,et al.Nanostructured materials in drug and gene delivery:a review of the state of the art[J].Crit Rev Ther Drug Carrier Syst,2011,28(2):101-164.
[25]Rezaee M,Oskuee RK,Nassirli H,et al.Progress in the development of lipopolyplexes as efficient non-viral gene delivery systems[J].J Control Release,2016,236(1):1-14.
[26]Jain AK,Massey A,Yusuf H,et al.Development of polymeric-cationic peptide composite nanoparticles,a nanoparticle-in-nanoparticle system for controlled gene delivery[J].Int J Nanomedicine,2015,10(1):7183-7196.
[2]王晓燕,袁中芳,魏增涛,等.重组IFN-l蛋白联合endostatin基因对碱烧伤诱导角膜新生血管的抑制作用[J].基础医学与临床,2005,25(12):1119-1123.WANG Xiaoyan,YUAN Zhongfang,WEI Zengtao,et al.Inhibitory effect of recombinant IFN-αcombined w ith endostatin gene on corneal neovascurization induced by alkaili burn[J].Basic and Clinical M edicine,2005,25(12):1119-1123.
[3]Mohajeri A,Pilehvar-Soltanahmadi Y,PourhassanM oghaddam M,et al.Cloning and expression of recombinant human endostatin in periplasm of Escherichia coli expression system[J].Adv Pharm Bull,2016,6(2):187-194.
[4]Tan H,Yang S,Feng Y,et al.Characterization and secondary structure analysis of endostatin covalently modified by polyethylene glycol and low molecular w eight heparin[J].J Biochem,2008,144(2):207-213.
[5]Fu W,Zhuo J,and Hu L.Differential effects of recombinant human endostatin treatment on differentiated and undifferentiated blood vessels in Lew is lung cancer[J].Oncol Lett,2017,13(1):196-200.
[6]Zhang X,Li Y,Cheng Y,et al.Tat PTD-endostatin:a novel anti-angiogenesis protein w ith ocular barrier permeability via eye-drops[J].Biochim Biophys Acta,2015,1850(6):1140-1149.
[7]Li Y,Li L,Li Z,et al.Tat PTD-Endostatin-RGD:a novel protein w ith anti-angiogenesis effect in retina via eye drops[J].Biochim Biophys Acta,2016,1860(10):2137-2147.
[8]张新科.穿膜肽Tat PTD介导的内皮抑素穿透眼球屏障及对眼部血管增生抑制作用的研究[D].济南:山东大学,2012.ZHANG Xinke.Study on the penetration of eye barrier and inhibition of ocular angiogenesis of endostatin w ith the mediation of Tat PTD[D].Jinan:Shandong University,2012.
[9]Campochiaro PA,Lauer AK,Sohn EH,et al.Lentiviral vector gene transfer of endostatin/angiostatin for macular degeneration(GEM)study[J].Hum Gene Ther,2017,28(1):99-111.
[10]Ojea-Jiménez I,Tort O,Lorenzo J,et al.Engineered nonviral nanocarriers for intracellular gene delivery applications[J].Biomed M ater,2012,7(5):054106.doi:10.1088/1748-6041/7/5/054106.
[11]Wang JJ,Zeng ZW,Xiao RZ,et al.Recent advances of chitosan nanoparticles as drug carriers[J].Int J Nanomedicine,2011,(6):765-774.
[12]Veilleux D,Nelea M,Biniecki K,et al.Preparation ofconcentrated chitosan/DNA nanoparticle formulations by lyophilization for gene delivery at clinically relevant dosages[J].J Pharm Sci,2016,105(1):88-96.
[13]Ramesan RM,Sharma CP.Modification of chitosan nanoparticles for improved gene delivery[J].Nanomedicine,2012,7(1):5-8.
[14]姚金凤,郭晨阳,杨红,等.壳聚糖纳米粒在药物输送中的应用研究进展[J].国际药学研究杂志,2013,40(1):90-94.YAO Jinfeng,GUO Chenyang,YANG Hong,et al.Application of chitosan nanoparicles for drug delivery:research advances[J].J Int Pharm Res,2013,40(1):90-94.
[15]Babu A,Ramesh R.Multifaceted applications of chitosan in cancer drug delivery and therapy[J].M ar Drugs,2017,15(4):96-104.
[16]Zhang X,Yang X,Ji J,et al.Tumor targeting strategies for chitosan-based nanoparticles[J].Colloids Surf B Biointerfaces,2016,148(1):460-473.
[17]Key J,Park K.Multicomponent,tumor-homing chitosan nanoparticles for cancer imaging and therapy[J].Int J M ol Sci,2017,18(3):594-612.
[18]Jung SP,Siegrist B,Hornick CA,et al.Effect of human recombinant endostatin protein on human angiogenesis[J].Angiogenesis,2002,5(1-2):111-118.
[19]Chen J,Liu DG,Yang G,et al.Endostar,a novel human recombinant endostatin,attenuates liver fibrosis in CCl4-induced mice[J].Exp Biol M ed,2014,239(8):998-1006.
[20]Ling Y,Yang Y,Lu N,et al.Endostar,a novel recombinant human endostatin,exerts antiangiogenic effect via blocking VEGF-induced tyrosine phosphorylation of KDR/Flk-1 of endothelial cells[J].Biochem Biophys Res Commun,2007,361(1):79-84.
[21]Xing P,Zhang J,Yan Z,et al.Recombined humanized endostatin(Endostar)combined w ith chemotherapy for advanced bone and soft tissue sarcomas in stage IV[J].Oncotarget,2017,8(22):36716-36727.
[22]Wigler M,Silverstein S,Lee LS,et al.Transfer of purified herpes virus thymidine kinase gene to cultured mouse cells[J].Cell,1977,11(1):223-232.
[23]张艳,马春红,孙汶生,等.高生物安全性人内皮抑素载体抑瘤作用的研究[J].中国现代普通外科进展,2004,7(6):350-355.ZHANG Yan,M A Chunhong,SUN Wensheng,et al.The antitumor effect of the high bio-safety vector containing human endosatin[J].Chin J Curr Adv Gen Surg,2004,7(6):350-355.
[24]Petkar KC,Chavhan SS,Agatonovik-Kustrin S,et al.Nanostructured materials in drug and gene delivery:a review of the state of the art[J].Crit Rev Ther Drug Carrier Syst,2011,28(2):101-164.
[25]Rezaee M,Oskuee RK,Nassirli H,et al.Progress in the development of lipopolyplexes as efficient non-viral gene delivery systems[J].J Control Release,2016,236(1):1-14.
[26]Jain AK,Massey A,Yusuf H,et al.Development of polymeric-cationic peptide composite nanoparticles,a nanoparticle-in-nanoparticle system for controlled gene delivery[J].Int J Nanomedicine,2015,10(1):7183-7196.